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Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
Chapter 19
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Chapter 19

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  • 1. Chapter 19 Spectrophotometry :Instruments & Application
  • 2. 19.1 The Spectrophotometer –1Remote sensing of airborne bacteria: Optical fiber coated with antibodies to detect spores of a specific bacterium
  • 3. 19.1 The Spectrophotometer -11) Spectrophotometerb) Single-beame) Double-beam
  • 4. 19.1 The Spectrophotometer -2
  • 5. 19.1 The Spectrophotometer -3
  • 6. 19.1 The Spectrophotometer –4• Light source – Tungsten lamp: Vis. near IR (320 nm~2500 nm) – Deuterium are lamp: UV (200~400 nm) – Electric discharge lamp + Hg(g) or Xenon: Vis & UV – Globar (silicon carbide rod): IR (5000~200 cm-1) – Laser: intense monochromatic sources.
  • 7. 19.1 The Spectrophotometer -5
  • 8. 19.1 The Spectrophotometer -6• MonochromatorConsists:• lenses or mirrors: focus the radiation• entrance and exit slits: restrict unwanted and control the spectral purity of radiation.• dispersing medium: separate the λ of polychromatic radiation from the source. (a) prism and (b) diffraction grating see Fig 19-2
  • 9. 19.1 The Spectrophotometer -71) Monochromator a. entrance slit b. collimating mirror or lens c. a prism or grating d. focal plane e. exit slit
  • 10. 19.1 The Spectrophotometer -8• Monochromator Interference of adjacent waves that are (a) 0°, (b) 90 °, and © 180 ° out of phase
  • 11. 19.1 The Spectrophotometer -9• Monochromator nλ = a – b (n = ±1 first-order….) Grating equation : nλ = d (sinθ – sinφ ) Filters: select a desired wavelength
  • 12. 19.1 The Spectrophotometer -10• MonochromatorChoosing the bandwidth: exit slit widthResolution trade-off Signal
  • 13. 19.1 The Spectrophotometer -101) Detector Convert radiant energy (photons) into an electrical signal Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.
  • 14. 19.1 The Spectrophotometer -11 3) Detector Detector response depends on the λ of the incident photons.
  • 15. 19.1 The Spectrophotometer -12Photomultiplier tube: very sensitive detector
  • 16. 19.1 The Spectrophotometer -13Photodiode array spectrophotometer :records the entire spectrum at once. vs. Dispersive spectrophotometer: one λ at a time • speed (~1s/spetrum) • excellent λ repeatability • measure λs simultaneously • relatively insensitive to errors from stray light • relatively poor resolution (1~3 nm) vs 0.1nm
  • 17. 19.1 The Spectrophotometer -14 diode array spectrophotometer
  • 18. 19.2 Analysis of a mixture -1• Absorbance of a mixture : A = exb[X] + eyb[Y] + …
  • 19. 19.2 Analysis of a mixture -2• Isosbestic points : for rxn: X → Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn. Ex: HIn  In- + H+
  • 20. 19.2 Analysis of a mixture -3Why isosbestic point? A 465 = ε 465 [ HIn ] HIn [ ] A 465 = ε 465 In − In − when [ HIn ] = [ In ] ⇒ ε − 465 HIn = ε 465 = ε 465 In − ∴ For a mixture : HIn In − [ ] A 465 = ε 465 b [ HIn ] + ε 465 b In − = ε 465 b ( [ In ] + [ HIn ] ) − −
  • 21. 19.3 Spectrophotometric Titrations -1 apotransferrin + 2Fe3+  (Fe3+)2transferrin colorless red (465 nm)
  • 22. 19.3 Spectrophotometric Titrations-2 Ferric nitrilotriacetate [used to avoid Fe(OH)3 ]
  • 23. 19.3 Spectrophotometric Titrations-3 ex.at p.408 Correcting A for the effect of dilution 125 μL ferric nitrilotriancetate 2 mL apotransferrin A = 0.260 A corrected = ?
  • 24. 19.4 What happens when a molecule absorbs light ? 1) Absorbing species : M + hν → M* (lifetime : 10-8 ~ 10-9 sec) Relaxation processes : • M* → M + heat (most common) • M* → new species (photochemical reaction) • M* → M + hν (fluorescence, phosphorescence)
  • 25. 19.4• Geometry of formaldehyde
  • 26. 19.4 What happens when a molecule absorbs light ? • Types of absorbing electrons Consider formaldehyde: three types of molecular orbitals • =σ H C O ×=π H =n
  • 27. 19.4 What happens absorbs light ? MO of CH3CO
  • 28. 19.4 What happens when a molecule absorbs light ? Four types of electronic transitions σ* π* E n 200~700 nm π 150~250 nm σ < 125 nm
  • 29. 19.4 What happens when a molecule absorbs light ? -5 1) Singlet / Triplet excited states ground excited excited singlet state singlet (S1) triplet (T1) E: T1 < S1
  • 30. 19.4 What happens when a molecule absorbs light ? -61) Electronic transition of formaldehyden → π* (T1), absorption of light at λ = 397 nm green-yellown → π* (S1), absorption of light at λ = 355 nm colorless (more probable)
  • 31. 19.4 What happens when absorbs light ?• Vibrational & Rotational states of CH3CO (IR and microwave radiation)
  • 32. 19.4 a molecule absorbs light 1) What happens to absorbed energy
  • 33. 19.4 a molecule absorbs light 7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis.  Photoluminescence : • Fluorescence : S1 → S0, no change in electron spin. (< 10-5 s) • Phosphorescence : T1 → S0, with a change in electron spin. (10-4~102 s) b. Chemiluminescence : Chemical reaction (not initiated by light) release energy in the form of light. ex : firefly.
  • 34. 19.4 a molecule absorbs light 1) In which your class really shines ? emission spectrum
  • 35. 19.4 a molecule absorbs light1) Absorption & Emission Spectra
  • 36. 19.5 Luminescence in analytical chemistry1) Instrument • .hνout (photon) • heat hνin • breaking a chemical bond
  • 37. 19.5 Luminescence• I = kPoC  incident radiation sensitivity  by P0  or C 6) more sensitive than Absorption
  • 38. 19.5 Luminescence4) Fluorimetric Assay of Selenium in Brazil Nuts – Se is a trace element essential to life: destruct ROOH (free radical) – Derivatized: – Self-absorption: quench
  • 39. 19.5 Luminescence5) Immunoassarys  employ anitbody to detect analyte. Ex: ELISA
  • 40. 19.5 Luminescence• pregnancy test. sensitive to < 1 ng of analyte• Enviromental Analysis. (ppm) or (ppt) pesticides, industrial chemicals, & microbialtoxins.
  • 41. 19.5 Luminescence• Environmental Analysis
  • 42. 19-14
  • 43. 19-15
  • 44. 19-18
  • 45. 19-21
  • 46. 19-22

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